TRANSCRIPTIONAL UP-REGULATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE GENE-EXPRESSION IN MELPHALAN-RESISTANT HUMAN PROSTATE CARCINOMA-CELLS

Tumor cell resistance to many chemotherapeutic agents, including alkyl ating agents, cisplatin, and doxorubicin, is frequently associated wit h increased intracellular levels of the nonprotein sulfhydryl glutathi one (GSH). Recent evidence has demonstrated that increased GSH levels can be accompanied by an increase in the activity of gamma-glutamylcys teine synthetase (GCS), which catalyzes the rate-limiting step in de n ovo synthesis of GSH, and by an increase in the steady state level of mRNA for the catalytic subunit of GCS. Using melphalan-resistant DU 14 5/M4.5 human prostate carcinoma cells, which express elevated GSH leve ls, GCS enzyme activity, and GCS mRNA levels, we sought to determine t he mechanism(s) responsible for the increased GCS mRNA expression. As determined by Northern analyses and RNase protection assays, the stead y state level of GCS message in the resistant cells was increased 10-2 0-fold, in comparison with the drug-sensitive parent DU 145 cells. No significant difference in gene copy number or evidence of rearrangemen t was detected in the resistant cell line by Southern analyses. The GC S-specific mRNA isolated from the resistant cells was less stable than that isolated from the drug-sensitive cells (half-lives of 6 hr and 9 hr, respectively), indicating that this difference does not contribut e to the increased steady state levels in the resistant cells. Nuclear run-on experiments revealed that the GCS transcription rate in the DU 145/M4.5 cells was increased approximately 12-fold, in comparison wit h that detected in the DU 145 cells. This difference in transcription rate was comparable in magnitude to the difference in steady state mRN A levels detectable in the two cell populations. Similar correlations between steady state GCS mRNA levels and transcription rates were also observed in other DU 145 lines expressing intermediate degrees of res istance to melphalan and correspondingly intermediate GCS mRNA elevati ons. These data suggest that GCS expression is transcriptionally regul ated in these melphalan-resistant tumor cells.